Association between SARS‐CoV‐2 infections during pregnancy and preterm live birth

Abstract We examined associations between mild or asymptomatic prenatal SARS‐CoV‐2 infection and preterm live birth in a prospective cohort study. During August 2020–October 2021, pregnant persons were followed with systematic surveillance for RT‐PCR or serologically confirmed SARS‐CoV‐2 infection until pregnancy end. The association between prenatal SARS‐CoV‐2 infection and preterm birth was assessed using Cox proportional‐hazards regression. Among 954 pregnant persons with a live birth, 185 (19%) had prenatal SARS‐CoV‐2 infection and 123 (13%) had preterm birth. The adjusted hazard ratio for the association between SARS‐CoV‐2 infection and preterm birth was 1.28 (95% confidence interval 0.82–1.99, p = 0.28), although results did not reach statistical significance.


| BACKGROUND
More than 225,000 cases of COVID-19 in pregnant persons were reported to the Centers for Disease Control and Prevention (CDC) during January 2020-July 2022. 1 The cumulative number of SARS-CoV-2 infections among pregnant persons in the United States is likely higher because some infections are asymptomatic or may not prompt clinical testing for laboratory confirmation.Although case series and surveillance studies of COVID-19 among pregnant persons suggest that most infections are mild, some studies have reported associations between prenatal COVID-19 and adverse pregnancy outcomes, including preterm live birth and stillbirth. 2,3However, studies examining the effect of prenatal SARS-CoV-2 infection have focused on medically attended infections that may not represent the majority of infections in the community, which are mild.Cross-sectional or retrospective study designs are subject to misclassification of infection status and recall error, while case-series do not include comparison with pregnant persons without infection.Understanding the impact of SARS-CoV-2 infection on pregnancy outcomes is important for informing guidance for pregnant persons and for characterizing the overall impact of COVID-19.To examine possible associations between prenatal SARS-CoV-2 infection and risk of preterm live birth, we used data from a multisite community cohort of pregnant persons who participated in prospective systematic surveillance for symptomatic and asymptomatic SARS-CoV-2 infections before the SARS-CoV-2 Omicron variants emerged.

| Study design and participants
During August 2020-February 2021, the Epidemiology of SARS-CoV-2 in Pregnancy and Infancy (ESPI) study enrolled a cohort of pregnant women at <28 weeks gestation at three US medical centers in Salt Lake City, UT; New York City, NY; and Birmingham, AL.Cohort participants were followed with active surveillance for SARS-CoV-2 infections through the end of their pregnancies; the follow-up period spanned August 2020-October 2021.
Detailed eligibility criteria and data collection procedures were previously described. 4At enrollment, participants completed self-administered electronic questionnaires about their medical and pregnancy histories including whether they were diagnosed with COVID-19 with a positive laboratory test during pregnancy before study enrollment.
From enrollment through the end of pregnancy, participants were asked to self-collect mid-turbinate nasal swab (MTS) specimens every week during pregnancy and ship specimens on ice packs by overnight courier to a central laboratory for reverse-transcription polymerase chain reaction (RT-PCR) testing for SARS-CoV-2.Participants also collected and shipped additional swab specimens if they experienced onset of COVID-like illness (CLI) symptoms, defined as ≥1 of measured or subjective fever, cough, shortness of breath, sore throat, diarrhea, muscle aches, chills, or change in taste or smell.In addition, participants had serum collected at enrollment, around 26-30 weeks of gestation (if enrolled at <24 weeks gestation) and at end of pregnancy; sera were tested for antibodies to SARS-CoV-2 as evidence of infection.
At the end of pregnancy, participants completed self-administered questionnaires about pregnancy outcomes and COVID-19 vaccine receipt during pregnancy.Pregnancy outcome and COVID-19 vaccination data were also abstracted from medical records.

| Ethical review
The study protocol was reviewed and approved by the Columbia University Irving Medical Center Institutional Review Board (IRB), which served as the single IRB for all study sites.The CDC IRB relied on the review of the Columbia University Irving Medical Center IRB.

| Laboratory methods
Respiratory swab specimens were tested by RT-PCR for SARS-CoV-2 using the Quidel Lyra SARS-CoV-2 Assay or the ThermoFisher Combo Kit platform with ThermoFisher probes and primers. 5

| Study definitions
The exposure of interest was SARS-CoV-2 infection during pregnancy defined as ≥1 of a self-reported laboratory-confirmed diag- The outcome of interest was preterm live birth, which was identified by an International Classification of Disease-10 (ICD-10) discharge diagnosis code of O60.1 or P07 in the delivery hospitalization medical record or defined as a live birth at gestational age <37 0/7 weeks calculated from participant-reported estimated delivery date if medical record data were not available.

| Sample size considerations
The goal sample size of the ESPI cohort was chosen to achieve a desired precision for calculating the cumulative incidence of SARS-CoV-2 infection.A priori sample size calculations for assessing the effect of SARS-CoV-2 on preterm birth were performing for varying effect sizes to identify the minimum effect size that could be detected with the predetermined cohort sample size.Assuming a preterm birth prevalence of 10% among persons without SARS-CoV-2 and a cumulative incidence of SARS-CoV-2 infection of ≥10%, a sample size of 1182 was determined to provide sufficient statistical power (with type I error of 5% and type II error of 20%) to detect a minimum effect size of prenatal SARS-CoV-2 exposure of 2.0.

| Statistical analysis
The analysis was restricted to participants who contributed to surveillance for SARS-CoV-2 by submitting ≥1 MTS sample during the study period.Participants were excluded if they had an induced abortion, reported being diagnosed with COVID-19 before pregnancy, or were seropositive at enrollment without a previous diagnosis of COVID-19 during pregnancy because of uncertainty about whether their infection occurred before or during pregnancy.Participants with pregnancy losses (miscarriages or stillbirths) were also excluded.
The association between SARS-CoV-2 infection during pregnancy and perinatal outcomes was assessed using Cox proportionalhazards regression.The time scale was weeks with week 0 defined as starting on the calculated date of conception.Participants who were lost to follow-up or withdrawn before end of pregnancy without data collection about pregnancy outcomes were included in models and censored at last surveillance respiratory specimen collection.Full-term pregnancies exited the risk-set after 36 weeks gestation.SARS-CoV-2 infection was treated as a time-varying covariate where a participant was considered exposed from the date of laboratory confirmation of their first infection.For participant-reported infections before study enrollment, participant-reported date of lab-

| RESULTS
During the study period, 1176 pregnant persons were enrolled and participated in surveillance for SARS-CoV-2 infection (Figure S1).Of these 1176 participants, 222 were excluded (three for induced abortions, 34 for diagnosis of COVID-19 before their pregnancy, 169 for seropositivity at enrollment without COVID-19 diagnosis during pregnancy, and 16 for pregnancy losses).Of the remaining 954 participants, 905 (95%) were followed through the end of pregnancy and 49 (5%) were lost to follow-up or withdrew without data collection on pregnancy outcomes.Participants in this analysis contributed a median of 13 person-weeks of observation time with weekly MTS submission and a median of 16 person-weeks of observation time once missing weeks were imputed using serology data.

| DISCUSSION
Using data from a prospective cohort study of >900 pregnant persons followed during early epidemic waves of SARS-CoV-2 circulation in the United States, we evaluated the association between largely mild or asymptomatic SARS-CoV-2 infection during pregnancy and preterm live birth.Almost one in five persons in this analysis had evidence of SARS-CoV-2 infection during pregnancy, and infection incidence was likely higher given that some persons with serologic Based-on questionnaires that asked about the following medical conditions: asthma, chronic lung disease other than asthma, chronic metabolic diseases such as diabetes mellitus types 1 and 2 and thyroid disease, blood disorders such as thalassemia and sickle cell disease, hypertension, cardiovascular disease, bladder/kidney disease, liver disease, immunocompromising conditions, neurologic/neuromuscular disease, and rheumatologic conditions.d Includes COVID-19 vaccination status before enrollment and during study follow-up.
evidence of infection at cohort enrollment were excluded from the analysis.We found no significant association between preterm birth among pregnant persons with prenatal SARS-CoV-2 infection compared with those without infection.
Some previous studies have found a significant association between SARS-CoV-2 infection and preterm birth (adjusted relative risk or odds ratio range: 1.2-1.8). 2,6,7Infections in our analysis were largely mild or asymptomatic and detected through systematic study testing, whereas these prior studies either focused on severe infections requiring hospitalization and/or infection status determined by clinical testing 8 that may be influenced by testing biases. 9More severe SARS-CoV-2 infections may be associated with more robust inflammatory responses that may adversely affect the placenta 10 and intrauterine environment and predispose to preterm birth.To prevent severe SARS-CoV-2 infections that may be associated with additional complications, COVID-19 vaccination for pregnant persons remains important and is recommended by CDC 11 and the American Society of Obstetrics and Gynecology. 12is analysis has several limitations.First, the sample size was small compared with some surveillance and registry studies, and the study was underpowered to detect an effect size unless it was relatively large.Despite this limitation, this study includes prospectively collected data from a cohort followed with rigorous systematic surveillance for SARS-CoV-2 infections and pregnancy outcomes that may contribute to meta-analyses examining the association between SARS-CoV-2 infections and pregnancy outcomes.Second, this study was conducted before the emergence of Omicron variant viruses, and respiratory samples that were positive for SARS-CoV-2 were not sequenced to identify virus variants.A previous study suggested that prenatal SARS-CoV-2 infection effects on perinatal outcomes may vary by variant type 8 underscoring the need to assess potential effects on birth outcomes as SARS-CoV-2 continues to circulate.
T A B L E 2 Crude and adjusted Hazard ratios of preterm live birth among pregnant individuals with versus without prenatal SARS-CoV-2 infection, N = 954.
nosis of COVID-19 during pregnancy before study enrollment, RT-PCR-confirmed infection detected by study surveillance, or serologic evidence of SARS-CoV-2 infection during the study.Participants were considered to have serologic evidence of SARS-CoV-2 infection if they had a negative serum sample followed by a positive serum sample indicating seroconversion.Serum samples were considered positive if the spike (S1) or RBD antigens were detected in samples collected before COVID-19 vaccination or if the N antigen was detected in samples collected after COVID-19 vaccination.
oratory confirmation was used to assign infection timing.For study weeks when participants did not submit weekly MTS, infection status was imputed using study serology results.Participants who were seronegative during all serum collection timepoints without RT-PCR-confirmed SARS-CoV-2 infection were assumed to be uninfected.Participants who seroconverted during the study without RT-PCR-confirmed SARS-CoV-2 infection were assumed to have been infected during the interval when they seroconverted, and their infection timing was assigned as the midpoint between the seronegative sample and 4 weeks before the first seropositive sample.The following variables were considered for inclusion in multivariable model: study site, age group (18-24, 25-34, and 35-50 years), highest educational level (less than college, some college or higher), presence of ≥1 chronic condition, self-reported smoking or alcohol use during pregnancy, multiple gestation pregnancy, diagnosis of gestational diabetes or gestational hypertension, and selfreport of ≥1 psychosocial stressor during pregnancy based on a 17 question series administered at the end of pregnancy.Study site and age group were chosen a priori for inclusion in all final models.Additional covariates with a p value <0.1 in the initial model were retained.The final model for preterm live birth included study site, age group, highest educational level, presence of ≥1 chronic condition, diagnosis of gestational diabetes, diagnosis of gestational hypertension, current smoking status, and multiple gestation pregnancy.A Poisson regression model that does not address gestational time as a potential confounder was also run as a sensitivity analysis to estimate relative risks for comparison with published estimates from other studies.Analyses were done in SAS version 9.4.

1
Baseline characteristics of cohort participants, N = 954.p values were calculated based on a Chi-square test unless a cell frequency <5, in which case a Fisher exact test was used.
b c Note: Adjusted Hazard Ratio estimate for preterm live birth was derived from a Cox proportional hazards model after adjusting for site, age group (18-24, 25-34, and 35-50 years), highest educational level, any chronic conditions, gestational diabetes, gestational hypertension, current smoking status, and multiple gestation.